Is SIX1 an effector in the Fusarium oxysporum f.sp … · 2017. 2. 9. · 1)University of...
1
1)University of Amsterdam, Molecular Plant Pathology, Science Park 904, 1098XH Amsterdam, NL 2)USDA-ARS Cereal Disease Laboratory 1551, Lindig Street, 55108 Saint Paul, MN 3)Plant Research Institute, Wageningen University of Research, Droevendaalsesteg 1, 6708 PB, Wageningen, NL Sri Widinugraheni 1) , Jonathan Niño Sánchez 2) , Lotje van der Does 1) , Fernando Garcia BasAdas 3) , Nadia Ordonez 3) , Gert Kema 3) , Corby Kistler 2) , and MarAjn Rep 1) Introduction MQ S.widinugraheni @ uva.nl Foc effector candidates SIX1c and possibly SIX1a can be recognized by the I-3 resistance gene of tomato The Foc six1a knock out mutant is compromised in virulence on banana Outlook RNA sequencing to identify more candidate effectors Gene knock-out and complementation of the candidate effectors Develop marker specific to a particular race of Foc We are thankful to Cees Waalwijk (PRI) &UNIFARM for providing facility for and assistance with the green house experiment at WUR, Wageningen. We also thank Altus Viljoen, Stellenbosch University (SUN), South Africa for providing Foc isolates. Plant weight (g) Plant weight (g) RIGHT: Several different transformants of Fol + FocSIX1a,b,c were generated. To test their pathogenicity those transformants were inoculated on I‐3 tomato and also on suscepCble tomato. The wild type strain (WT), the Δsix1 strain and water were also included as a control. IniCal results show that SIX1c and possibly SIX1a can complement for avirulence, indicated by higher fresh weight on I‐3 plants accompanied by lower disease index. Blue circles indicate the control strains, the red circle indicates the transformants unrecognized by I‐3, and the green circle indicates transformants that are recognized by I‐3. Transformants that show reduced virulence on the suscepCble plant line are indicated by purple circle. Each dot is the average of 15 plants. DI = Disease Index 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% water WT KO DI=3 DI=2 DI=1 DI=0 DI=0, if length of vessel browning in corm 0 – 0.5 cm ; DI=1, if 0.5cm <length of vessel browning in corm ≤ 1cm ; DI=2, if 1cm <length of vessel browning in corm ≤ 2cm; DI=3, if 2cm <length of vessel browning in corm ≤ 3cm DI=0, if % of yellowing, wil‘ng and necro‘c leaves = 0 % DI=1, if % of yellowing, wil‘ng and necro‘c leaves ≤ 25% DI=2, if 25% ≤ % of yellowing, wil‘ng and necro‘c leaves ≤ 50% DI=3, if 50% ≤ % of yellowing, wil‘ng and necro‘c leaves ≤ 100% water Foc wild type Foc Δ six1a Visual symptoms were measured as disease index at 13 weeks aQer inoculaCon. -SPIN Project internal symptoms 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% water WT TR4 KO DI=3 DI=2 DI=1 DI=0 We inoculated suscep‘ble Cavendish cv. Grande Naine with Foc TR4 wild type strain II5 and a Foc Δsix1a mutant. We observed symptoms internally by looking at brown vessels in the corm and externally by looking at yellowing, necrosis and wil‘ng of the leaves. The Δsix1a strain produces less vessel browning than the wild type The Δsix1a strain results in fewer symptoms on leaves than the wild type (a) (b) Is SIX1 an effector in the Fusarium oxysporum f.sp cubense‐banana interacAon ? DI Acknowledgement: Fusarium oxysporum f.sp. cubense (Foc) is the causal agent of Panama disease in banana. Foc has evolved into several different races (right panel). In the Fusarium oxysporum f.sp. lycopersici (Fol)‐tomato system, small secreted proteins (Six, S ecreted i nx ylem) can act as virulence factors, but may also trigger resistance in the host. In tomato, three resistance genes are known, I‐1, I‐2 and I‐3, and the corresponding AVR genes SIX4(AVR1), SIX3(AVR2), and SIX1(AVR3) have been iden‘fied. In Foc, however, ligle is known about the effectors necessary to infect banana. We iden‘fied 11 candidate effectors in the genome sequence of a Foc TR4 strain (II5, sequenced and annotated by the Broad Ins‘tute) based on homology to SIX genes and other characteris‘cs. Here we present results of our ini‘al characteriza‘on of these candidate effector genes. Can Foc SIX1a,b or c be recognized by I‐3 of tomato? Transform a FolΔsix1 strain with FocSIX1a,b or c Inoculate transformants on suscep‘ble and I‐3 tomato Which effector genes have a virulence func‘on? Generate knock‐out strains Inoculate suscep‘ble banana cul‘var Research Pipeline Which effector genes differen‘ate Foc races or clonal lines? PCR effector candidates from a collec‘on of strains Determine sequence of effector genes I‐3 recogni‘on Less symptoms with KO mutant Restricted to clonal line or race AVR3 candidate virulence func‘on marker water 0.00 six1b 3.33 six1b 3.53 six1b 3.40 six1b 3.40 six1b 3.20 six1a 2.73 six1c 1.60 six1a 0.93 six1a 2.67 SIX1a 3.67 six1c 1.60 six1c 1.27 six1 c 1.29 six1c 1.33 six1c 0.47 Fol Δ SIX1 3.80 Fol WT 0.73 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 Disease Index and Plant Weight on I‐3 tomato Foc SIX1a, b and c appear to be restricted to the TR4 clonal line whereas Foc SIX9 and FocEf3 are present in all clonal lines group D (ST4) group A (TR4) group B (R1,ST4) group F (R1) group E group C (R1,R2) Conclusions Some of the FocSIX1 transformants are recognized by the I‐3 gene in tomato, indica‘ng that Six1 homologs from Foc can have an avirulence func‘on. The SIX1a knock‐out mutant showed less virulence, sugges‘ng that Six1a has a virulence func‘on during banana infec‘on. Foc SIX1a,b and c appear to be restricted to the TR4 clonal line. Six different TEF1a sequences were found in Foc isolates, assumed to represent clonal lines. The presence of candidate effectors in these clonal lines is shown to the right Foc Races Host Race 1 (R1) Banana var. Gros Michel, Lady Finger, Duccase, Sugar, Silk, but not Cavendish Race 2 (R2) Banana var. Bluggoe and Blue Java, but not Cavendish Race 3 Heliconia, not bananas Sub‐Tropical Race 4 (ST4) (under specific abio‘c condi‘on) Cavendish and other banana varie‘es Tropical Race 4 (TR4) Cavendish and other banana varie‘es IF THEN water 0.00 six1b 2.60 six1b 3.00 six1 b 2.87 six1c 2.67 six1b 2.60 six1a 2.33 six1a 2.40 six1a 2.93 six1a 2.00 six1a 2.33 six1c 2.67 six1 c 2.00 six1c 3.27 six1 b 2.67 six1c 2.67 Fol ΔSIX1 3.73 Fol WT 3.93 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 0.00 2.00 4.00 6.00 8.00 10.00 12.00 Disease Index and Plant Weight on suscep‘ble tomato SIX1a SIX1b SIX1c SIX6 SIX8 SIX9 SIX13 FocEf1 FocEf2 FocEf3 FocEf4 A A A A A A A A A A A A A A A A A A B absent some- times present always present sequence grouping within effectors DI
Transcript of Is SIX1 an effector in the Fusarium oxysporum f.sp … · 2017. 2. 9. · 1)University of...
1) University of Amsterdam, Molecular Plant Pathology, Science Park 904, 1098XH Amsterdam, NL 2) USDA-ARS Cereal Disease Laboratory 1551, Lindig Street, 55108 Saint Paul, MN 3) Plant Research Institute, Wageningen University of Research, Droevendaalsesteg 1, 6708 PB, Wageningen, NL
Sri Widinugraheni 1), Jonathan Niño Sánchez 2), Lotje van der Does 1), Fernando Garcia BasAdas 3), Nadia Ordonez 3), Gert Kema 3), Corby Kistler 2), and MarAjn Rep 1)
Introduction
MQ
Foc effector candidates SIX1c and possibly SIX1a can be recognized by the I-3 resistance gene of tomato
The Foc six1a knock out mutant is compromised in virulence on banana
Outlook RNA sequencing to identify more candidate effectors Gene knock-out and complementation of the candidate effectors Develop marker specific to a particular race of Foc
We are thankful to Cees Waalwijk (PRI) &UNIFARM for providing facility for and assistance with the green house experiment at WUR, Wageningen. We also thank Altus Viljoen, Stellenbosch University (SUN), South Africa for providing Foc isolates.
Plant weight (g) Plant weight (g)
RIGHT: Several different transformants of Fol + FocSIX1a,b,c were generated. To test their pathogenicity those transformants were inoculated on I‐3 tomato and also on suscepCble tomato. The wild type strain (WT), the Δsix1 strain and water were also included as a control. IniCal results show that SIX1c and possibly SIX1a can complement for avirulence, indicated by higher fresh weight on I‐3 plants accompanied by lower disease index. Blue circles indicate the control strains, the red circle indicates the transformants unrecognized by I‐3, and the green circle indicates transformants that are recognized by I‐3. Transformants that show reduced virulence on the suscepCble plant line are indicated by purple circle. Each dot is the average of 15 plants. DI = Disease Index
0% 10% 20% 30% 40% 50% 60% 70% 80% 90%
100%
water WT KO
DI=3
DI=2
DI=1
DI=0
DI=0, if length of vessel browning in corm 0 – 0.5 cm ; DI=1, if 0.5cm <length of vessel browning in corm ≤ 1cm ; DI=2, if 1cm <length of vessel browning in corm ≤ 2cm; DI=3, if 2cm <length of vessel browning in corm ≤ 3cm
DI=0, if % of yellowing, wil`ng and necro`c leaves = 0 % DI=1, if % of yellowing, wil`ng and necro`c leaves ≤ 25% DI=2, if 25% ≤ % of yellowing, wil`ng and necro`c leaves ≤ 50% DI=3, if 50% ≤ % of yellowing, wil`ng and necro`c leaves ≤ 100%
water Foc wild type Foc Δ six1a
Visual symptoms were measured as disease index at 13 weeks aQer inoculaCon.
-SPIN Project
internal symptom
s
0% 10% 20% 30% 40% 50% 60% 70% 80% 90%
100%
water WT TR4 KO
DI=3 DI=2 DI=1 DI=0
We inoculated suscep`ble Cavendish cv. Grande Naine with Foc TR4 wild type strain II5 and a Foc Δsix1a mutant. We observed symptoms internally by looking at brown vessels in the corm and externally by looking at yellowing, necrosis and wil`ng of the leaves.
The Δsix1a strain produces less vessel browning than the wild type
The Δsix1a strain results in fewer symptoms on leaves than the wild type
(a)
(b)
Is SIX1 an effector in the Fusarium oxysporum f.sp cubense‐banana interacAon ?
DI
Acknowledgement :
Fusarium oxysporum f.sp. cubense (Foc) is the causal agent of Panama disease in banana. Foc has evolved into several different races (right panel). In the Fusarium oxysporum f.sp. lycopersici (Fol)‐tomato system, small secreted proteins (Six, Secreted in xylem) can act as virulence factors, but may also trigger resistance in the host. In tomato, three resistance genes are known, I‐1, I‐2 and I‐3, and the corresponding AVR genes SIX4(AVR1), SIX3(AVR2), and SIX1(AVR3) have been iden`fied. In Foc, however, ligle is known about the effectors necessary to infect banana. We iden`fied 11 candidate effectors in the genome sequence of a Foc TR4 strain (II5, sequenced and annotated by the Broad Ins`tute) based on homology to SIX genes and other characteris`cs. Here we present results of our ini`al characteriza`on of these candidate effector genes.
Can Foc SIX1a,b or c be recognized by I‐3 of tomato?
Transform a FolΔsix1 strain with FocSIX1a,b or c
Inoculate transformants on suscep`ble and I‐3 tomato
Which effector genes have a virulence func`on?
Generate knock‐out strains
Inoculate suscep`ble banana cul`var
Research Pipeline
Which effector genes differen`ate Foc races or clonal lines?
PCR effector candidates from a collec`on of strains
Determine sequence of effector genes
I‐3 recogni`on
Less symptoms with KO mutant
Restricted to clonal
line or race
AVR3 candidate
virulence func`on
marker
water 0.00
six1b 3.33
six1b 3.53
six1b 3.40
six1b 3.40
six1b 3.20
six1a 2.73
six1c 1.60
six1a 0.93
six1a 2.67
SIX1a 3.67
six1c 1.60
six1c 1.27
six1 c 1.29 six1c
1.33
six1c 0.47
Fol Δ SIX1 3.80
Fol WT 0.73
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00
Disease Index and Plant Weight on I‐3 tomato
Foc SIX1a, b and c appear to be restricted to the TR4 clonal line whereas Foc SIX9 and FocEf3 are present in all clonal lines
group D (ST4)
group A (TR4)
group B (R1,ST4)
group F (R1)
group E
group C (R1,R2)
Conclusions Some of the FocSIX1 transformants are recognized by the I‐3 gene in tomato, indica`ng that Six1 homologs
from Foc can have an avirulence func`on.
The SIX1a knock‐out mutant showed less virulence, sugges`ng that Six1a has a virulence func`on during
banana infec`on.
Foc SIX1a,b and c appear to be restricted to the TR4 clonal line.
Six different TEF1a sequences were found in Foc isolates, assumed to represent clonal lines. The presence of candidate effectors in these clonal lines is shown to the right
Foc Races Host
Race 1 (R1) Banana var. Gros Michel, Lady Finger, Duccase, Sugar, Silk, but not Cavendish
Race 2 (R2) Banana var. Bluggoe and Blue Java, but not Cavendish
Race 3 Heliconia, not bananas
Sub‐Tropical Race 4 (ST4) (under specific abio`c condi`on)
Cavendish and other banana varie`es
Tropical Race 4 (TR4) Cavendish and other banana varie`es
IF THEN
water 0.00
six1b 2.60
six1b 3.00 six1 b
2.87
six1c 2.67
six1b 2.60
six1a 2.33
six1a 2.40
six1a 2.93
six1a 2.00
six1a 2.33
six1c 2.67
six1 c 2.00
six1c 3.27
six1 b 2.67
six1c 2.67
Fol ΔSIX1 3.73
Fol WT 3.93
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
0.00 2.00 4.00 6.00 8.00 10.00 12.00
Disease Index and Plant Weight on suscep`ble tomato
SIX1a SIX1b SIX1c SIX6 SIX8 SIX9 SIX13 FocEf1 FocEf2 FocEf3 FocEf4
A A A
A A A A
A A A
A A A
A A A
A A B
absent
some-times present
always present
sequencegrouping within effectors
DI
